Identification Of STAU2 As A Promotive Host Factor For AIV By Viral Host Dual RNA-seq

H5N1 Avian Influenza Virus(AIV)belongs to highly pathogenic influenza virus,posing a serious threat to poultry production and public health security.In this study,the dynamic regulation of virus and host gene expression in AIV infected chicken embryonic fibroblasts(DF1)was studied by using Dual RNA-seq technique.The main conclusions are as follows:1.After AIV infected with chicken DF1 cells for 0 h,6 h,12 h and 20 h,the expression level of different viral genes was different but all showed increasing trend,among which the expression level of polymerase gene PA,PB1 and PB2 was lower than that of other viral genes such as HA and NA.In this study,a total of 2,762 differentially expressed genes were identified between the uninfected group and the infected group.2.Pathway enrichment analysis showed that in the process of virus infection,Cytokine-cytokine receptor interaction pathway and Inflammation mediated by chemokine and cytokine signaling pathway were significantly enriched.It showed that cytokines and chemokines and their related signaling pathways played an important role in the process of viral infection.3.Comparative analysis of the results of Dual RNA-seq and IP-MS showed that among the differences of expressed genes caused by virus infection,130 genes were identified to have interaction with AIV,including CCT5 and CSE1 L,suggesting that these genes may play potential roles in virus infection and replication.4.STAU2 was significantly down-regulated after viral infection,main role of the gene was to promote the localization and transport of mRNA in different organelles,and this study took the gene as a candidate gene to carry out in-depth mechanism research.The results of immunoprecipitation showed that STAU2 protein interacts with NS1 protein.The results of Immunofluorescence showed that the two proteins showed obvious co-localization in the cytoplasm.Knocking down STAU2 resulted in significantly lower virus titer,indicating that STAU2 is a gene promoting the replication of AIV.The infected DF1 cells were then separated into nuclear and cytoplasmic fractions followed by RNA extraction of each part to detect the content distribution of NS1 mRNA in the nuclear and cytoplasmic fractions.The results showed that there was no significant difference of NS1 mRNA in total RNA extracted from siSTAU2 and control cells.However,the level of NS1 mRNA in siSTAU2 group was significantly lower than that in the control group in cytoplasm,but higher in nucleus,indicating that STAU2 could promote the NS1 mRNA export of H5N1 IAV from nucleus to cytoplasm.Knocking down STAU2,the apoptosis level was significantly upregulated,so it was speculated that STAU2 might also promote the replication of AIV by inhibiting the apoptosis during virus infection.To sum up,this study explored the dynamic changes of virus and host genes after H5N1 AIV infection with chicken cells by Dual RNA-seq technique,identified the pro-avian influenza virus gene STAU2,and analyzed its mechanism of promoting influenza virus replication by promoting nuclear export of virus NS1 mRNA and anti-apoptosis.